PT2352763E - Bispecific single chain antibodies with specificity for high molecular weight target antigens - Google Patents

Bispecific single chain antibodies with specificity for high molecular weight target antigens Download PDF

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PT2352763E
PT2352763E PT09736398T PT09736398T PT2352763E PT 2352763 E PT2352763 E PT 2352763E PT 09736398 T PT09736398 T PT 09736398T PT 09736398 T PT09736398 T PT 09736398T PT 2352763 E PT2352763 E PT 2352763E
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rdc
domain
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Kufer Peter
Blümel Claudia
Kischel Roman
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Amgen Res (Munich) Gmbh
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

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Claims (13)

REIVINDICAÇÕES 1. Processo para a seleção de anticorpos biespecíficos de cadeia única contendo um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 e a um segundo domínio de ligação capaz de se ligar ao domínio extracelular do antigénio da membrana específica da próstata (AMEP), caracterizado pelo facto de compreender as etapas de: (a) providenciar pelo menos três tipos de células hospedeiras gue expressam (i.) o domínio extracelular humano de tipo selvagem de AMEP tendo a seg ID: 447 na superfície celular; (ii) uma forma mutada do AMEP humano de tipo selvagem na superfície celular, em que os resíduos de aminoácidos, nas posições 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 e 721 estão mutados nos correspondentes resíduos de aminoácidos do AMEP de tipo selvagem de roedor; e (iii) o domínio extracelular de tipo selvagem de AMEP de roedor na superfície celular; (b) fazer contatar cada tipo das células hospedeiras de (i) , (ii) e (iii) da etapa (a) com os anticorpos biespecíficos de cadeia única e células T efetoras; e (c) identificar e isolar anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras que expressam o domínio extracelular humano de tipo selvagem de AMEP na superfície celular, de acordo com (b) (i) e das células hospedeiras que expressam a forma mutada do AMEP humano de tipo selvagem, na superfície celular, de acordo com (b) (ii) , mas não das células hospedeiras que expressam o domínio extracelular AMEP de tipo selvagem de roedor, na superfície celular, de acordo com b (iii) .A process for the selection of single chain bispecific antibodies containing a first binding domain capable of binding to a CD3 antigenic determinant and a second binding domain capable of binding to the extracellular domain of the prostate specific membrane antigen ( AMEP) comprising the steps of: (a) providing at least three types of host cells expressing (i.) The wild-type human AMEP wild-type extracellular domain having a cell surface ID: 447; (ii) a mutated form of wild-type human AMEP on the cell surface, wherein the amino acid residues at positions 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 and 721 are mutated to the corresponding amino acid residues of wild-type rodent AMEP; and (iii) the wild-type extracellular domain of rodent AMEP on the cell surface; (b) contacting each type of the host cells of (i), (ii) and (iii) of step (a) with the single chain bispecific antibodies and effector T cells; and (c) identifying and isolating single-chain bispecific antibodies that mediate the lysis of host cells expressing the human wild-type extracellular domain of AMEP on the cell surface according to (b) (i) and of the host cells expressing the mutated form of the wild-type human AMEP on the cell surface according to (b) (ii), but not the host cells expressing the extracellular wild-type rodent AMEP domain on the cell surface, according to (b) ). 2. Processo para a seleção de anticorpos biespecíficos de cadeia única contendo um primeiro domínio capaz de se ligar a um determinante antigénico de CD3 e um segundo domínio de ligação capaz de se ligar ao domínio extracelular da proteína de ativação de fibroblastos α (PAFa), caracterizado pelo facto de compreender as etapas de: (a) providenciar pelo menos três tipos de células hospedeiras que expressam (i) o domínio extracelular humano de tipo selvagem de PAFa tendo a seq ID: 448 na superfície celular; (ii) uma forma mutada do PAFa humana de tipo selvagem, na superfície celular, em que os resíduos de aminoácidos, nas posições 144, 185, 186, 229, 267, 273, 274, 278, 284, 301, 328, 329, 331, 335 e 362 estão mutados nos correspondentes resíduos de aminoácidos do PAFa de tipo selvagem de roedor; e (iii) o domínio extracelular de tipo selvagem de roedor de PAFa na superfície celular; (b) fazer contatar cada tipo de células hospedeiras (i) , (ii) e (iii) , da etapa (a) , com os anticorpos biespecíficos de cadeia única e células T efetoras; e (c) identificar e isolar os anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras que expressam o domínio extracelular humano de tipo selvagem de PAFa, na superfície celular, de acordo com (b) (i) e das células hospedeiras que expressam a forma mutada do PAFa humano de tipo selvagem, na superfície celular, de acordo com (b) (ii) , mas não das células hospedeiras que expressam o domínio extracelular de PAFa, de tipo selvagem, de roedor, PAFa na superfície celular, de acordo com b(iii).A process for the selection of single-chain bispecific antibodies containing a first domain capable of binding to a CD3 antigenic determinant and a second binding domain capable of binding to the extracellular domain of the α-fibroblast activation protein (PAFÎ ±), characterized in that it comprises the steps of: (a) providing at least three types of host cells expressing (i) the wild-type human extracellular domain of PAFÎ ± having the cell surface seq ID: 448; (ii) a mutated form of the wild-type, wild-type human PAFα on the cell surface, wherein the amino acid residues, positions 144, 185, 186, 229, 267, 273, 274, 278, 284, 301, 328, 329, 331, 335 and 362 are mutated into the corresponding amino acid residues of wild-type rodent PAFα; and (iii) wild-type extracellular rodent domain of PAFa on the cell surface; (b) contacting each type of host cells (i), (ii) and (iii) of step (a) with the single chain bispecific antibodies and effector T cells; and (c) identifying and isolating single-chain bispecific antibodies that mediate the lysis of host cells expressing the human wild-type extracellular domain of PAFÎ ± of PAFÎ ± on the cell surface according to (b) (i) and the host cells which express the mutated form of the wild-type human, cell-surface PAFα according to (b) (ii), but not of the host cells expressing the extracellular domain of PAFα, wild type, rodent, cell surface PAFα, according to b (iii). 3. Processo para a seleção de anticorpos biespecificos de cadeia única, caracterizado pelo facto de compreender um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 e um segundo domínio de ligação capaz de se ligar ao domínio extracelular do recetor do fator de crescimento de hepatócitos (c-MET), endosialina (TEMI) e do recetor do tipo 1 do fator de crescimento semelhante à insulina (Rl-FCI), compreendendo as etapas de: (a) identificação dos 640 aminoácidos da membrana proximal do homólogo humano e do homólogo de roedor do domínio de c-MET, TEMI ou Rl-FCI; (b) fornecimento de células hospedeiras que expressam (i )o tipo selvagem humano do domínio extracelular dos domínios extracelulares c-MET, TEMI ou Rl-FCI, na superfície celular; (ii) uma proteína de fusão que contém 640 resíduos de aminoácidos da membrana proximal humana identificados na etapa (a) e > 640 resíduos de aminoácidos de roedor de c-MET, TEMI ou Rl-FCI; e (iii) o domínio extracelular de tipo selvagem de roedor de c-MET, TEMI ou Rl-FCI; (c) fazer contatar as células hospedeiras, de acordo com a etapa (b) , com os anticorpo biespecífico de cadeia única e células T efetoras; e (d) identificação e isolamento dos anticorpos biespecíficos de cadeia única que medeiam a lise das células hospedeiras, de acordo com (b)(i) e (b)(ii), mas não as células hospedeiras de acordo com b(iii).A method for the selection of single chain bispecific antibodies comprising a first binding domain capable of binding to a CD3 antigenic determinant and a second binding domain capable of binding to the extracellular domain of the factor receptor (TEM) and insulin-like growth factor type 1 receptor (IGF-I), comprising the steps of: (a) identifying the 640 amino acids of the homologous proximal membrane human and the rodent homolog of the c-MET, TEMI or Rl-FCI domain; (b) providing host cells expressing (i) wild-type human extracellular domain of extracellular domains c-MET, TEMI or Rl-FCI, on the cell surface; (ii) a fusion protein containing 640 amino acid residues of the human proximal membrane identified in step (a) and> 640 rodent amino acid residues of c-MET, TEMI or R1-FCI; and (iii) the wild-type extracellular rodent domain of c-MET, TEMI or R1-FCI; (c) contacting the host cells according to step (b) with the single chain bispecific antibody and effector T cells; and (d) identifying and isolating single-chain bispecific antibodies that mediate lysis of host cells according to (b) (i) and (b) (ii), but not host cells according to (b) . 4. Processo de acordo com uma qualquer das reivindicações 1 a 3, caracterizado pelo facto de o primeiro domínio de ligação se ligar a CD3 épsilon (CD3s) de seres humanos e de Callithrix jacchus, Saguinus oedipus ou Saimiri sciureus.A method according to any one of claims 1 to 3, wherein the first binding domain binds to CD3 epsilon (CD3s) from humans and from Callithrix jacchus, Saguinus oedipus or Saimiri sciureus. 5. Processo, de acordo com a reivindicação 4, caracterizado pelo facto de o primeiro domínio de ligação compreender uma região VL que contém RDC-Ll, RDC-L2 e RDC-L3 selecionadas entre: (a) RDC-Ll tal como descrito na SEQ ID N2. 27, RDC-L2 tal como descrito na SEQ ID N2. 28, RDC-L3 tal como descrito na SEQ ID N2. 29; (b) RDC-Ll tal como descrito na SEQ ID N2. 117, RDC-L2 tal como descrito na SEQ ID N2. 118, RDC-L3 tal como descrito na SEQ ID N2. 119; e (c) RDC-Ll tal como descrito na SEQ ID N2. 153, RDC-L2 tal como descrito na SEQ ID N2. 154, RDC-L3 tal como descrito na SEQ ID N2. 155.A method according to claim 4, wherein the first binding domain comprises a VL region containing RDC-L1, RDC-L2 and RDC-L3 selected from: (a) RDC-L1 as described in SEQ ID NO: 2. 27, RDC-L2 as described in SEQ ID NO: 2. 28, RDC-L3 as described in SEQ ID NO: 2. 29; (b) RDC-L1 as described in SEQ ID NO: 2. 117, RDC-L2 as described in SEQ ID NO: 2. 118, RDC-L3 as described in SEQ ID NO: 2. 119; and (c) RDC-L1 as described in SEQ ID NO: 2. 153, RDC-L2 as described in SEQ ID NO: 2. 154, RDC-L3 as described in SEQ ID NO: 2. 155. 6. Processo, de acordo com a reivindicação 4, caracterizado pelo facto de o primeiro domínio de ligação compreender uma região VP que contém as RDC-P1, RDC-P2 e RDC-P3 selecionadas entre: (a) RDC-P1 tal como descrito na SEQ ID N2. 12, RDC-P2 tal como descrito na SEQ ID N2. 13 e RDC-P3 tal como descrito na SEQ ID N2. 14; (b) RDC-P1 tal como descrito na SEQ ID N2. 30, RDC-P2 tal como descrito na SEQ ID N2. 31 e RDC-P3 tal como descrito na SEQ ID N2. 32; (c) RDC-P1 tal como descrito na SEQ ID N2. 48, RDC-P2 tal como descrito na SEQ ID N2. 49 e RDC-P3 tal como descrito na SEQ ID N2. 50; (d) RDC-P1 tal como descrito na SEQ ID N2. 66, RDC-P2 tal como descrito na SEQ ID N2. 67 e RDC-P3 tal como descrito na SEQ ID N2. 68; (e) RDC-P1 tal como descrito na SEQ ID N2. 84, RDC-P2 tal como descrito na SEQ ID N2. 85 e RDC-P3 tal como descrito na SEQ ID N2. 86; (f) RDC-P1 tal como descrito na SEQ ID N2. 102, RDC-P2 tal como descrito na SEQ ID N2. 103 e RDC-P3 tal como descrito na SEQ ID N2. 104; (g) RDC-P1 tal como descrito na SEQ ID N2. 120, RDC-P2 tal como descrito na SEQ ID N2. 121 e RDC-P3 tal como descrito na SEQ ID N2. 122; (h) RDC-P1 tal como descrito na SEQ ID N2. 138, RDC-P2 tal como descrito na SEQ ID N2. 139 e RDC-P3 tal como descrito na SEQ ID N2. 140; (i) RDC-P1 tal como descrito na SEQ ID N2. 156, RDC-P2 tal como descrito na SEQ ID N2. 157 e RDC-P3 tal como descrito na SEQ ID N2. 158; e (j) RDC-P1 tal como descrito na SEQ ID N2. 174, RDC-P2 tal como descrito na SEQ ID N2. 175 e RDC-P3 tal como descrito na SEQ ID N2. 176.A method according to claim 4, wherein the first binding domain comprises a VP region containing the RDC-P1, RDC-P2 and RDC-P3 selected from: (a) RDC-P1 as described in SEQ ID NO: 2. 12, RDC-P2 as described in SEQ ID NO: 2. 13 and RDC-P3 as described in SEQ ID NO: 2. 14; (b) RDC-P1 as described in SEQ ID NO: 2. 30, RDC-P2 as described in SEQ ID NO: 2. 31 and RDC-P3 as described in SEQ ID NO: 2. 32; (c) RDC-P1 as described in SEQ ID NO: 2. 48, RDC-P2 as described in SEQ ID NO: 2. 49 and RDC-P3 as described in SEQ ID NO: 2. 50; (d) RDC-P1 as described in SEQ ID NO: 2. 66, RDC-P2 as described in SEQ ID NO: 2. 67 and RDC-P3 as described in SEQ ID NO: 2. 68; (e) RDC-P1 as described in SEQ ID NO: 2. 84, RDC-P2 as described in SEQ ID NO: 2. 85 and RDC-P3 as described in SEQ ID NO: 2. 86; (f) RDC-P1 as described in SEQ ID NO: 2. 102, RDC-P2 as described in SEQ ID NO: 2. 103 and RDC-P3 as described in SEQ ID NO: 2. 104; (g) RDC-P1 as described in SEQ ID NO: 2. 120, RDC-P2 as described in SEQ ID NO: 2. 121 and RDC-P3 as described in SEQ ID NO: 2. 122; (h) RDC-P1 as described in SEQ ID NO: 2. 138, RDC-P2 as described in SEQ ID NO: 2. 139 and RDC-P3 as described in SEQ ID NO: 2. 140; (i) RDC-P1 as described in SEQ ID NO: 2. 156, RDC-P2 as described in SEQ ID NO: 2. 157 and RDC-P3 as described in SEQ ID NO: 2. 158; and (j) RDC-P1 as described in SEQ ID NO: 2. 174, RDC-P2 as described in SEQ ID NO: 2. 175 and RDC-P3 as described in SEQ ID NO: 2. 176. 7. Processo, de acordo com uma qualquer das reivindicações 3 a 6, caracterizado pelo facto de o segundo domínio de ligação se ligar aos determinantes antigénicos/sítios de ligação selecionados no grupo que consiste em: determinantes antigénicos/sítios de ligação nos quatro domínios de Ig com a seq ID: 436, um domínio rico em cisteína com a seq ID:437 ou a cadeia beta de um domínio sema com a seq ID: 438 do domínio extracelular de c-MET, determinantes antigénicos/sítios de ligação no domínio de mucino com a seq ID: 440, os três domínios semelhantes a FCE com a seq ID: 441 ou o domínio Sushi/SCR/CCP com a seq ID: 442 do domínio extracelular de TEMI e determinantes antigénicos/sítios de ligação nos três domínios de fibronectin do tipo III com a seq ID: 444 e o domínio L2 com a seq ID: 445 do domínio extracelular de Rl-FCI.A method according to any one of claims 3 to 6, wherein the second binding domain binds to the antigenic determinants / binding sites selected from the group consisting of: antigenic determinants / binding sites in the four domains of Ig with seq ID: 436, a cysteine-rich domain having the sequence ID: 437 or the beta chain of a sema domain with the c-MET extracellular domain seq ID: 438, antigenic determinants / binding sites in the domain of 4, the three CSF-like domains with SEQ ID: 441 or the Sushi / SCR / CCP domain with the sequence ID: 442 of the TEMI extracellular domain and antigenic determinants / binding sites in the three domains of fibronectin type III with seq ID: 444 and domain L2 with seq ID: 445 of the extracellular domain of R 1 -IFF. 8. Anticorpo biespecífico de cadeia única compreendendo um primeiro domínio de ligação capaz de se ligar a um determinante antigénico de CD3 épsilon (CD3s) de seres humano e primatas não chimpanzés, em que o determinante antigénico faz parte de uma sequência de aminoácidos compreendida no grupo que consiste nas SEQ ID N2s. 2, 4, 6 e 8 e um segundo domínio de ligação selecionado no grupo que consiste em: (a) um domínio de ligação capaz de se ligar ao domínio extracelular de AMEP humano mutado com uma sequência de aminoácidos tal como a representada na SEQ ID N2 . 447, em que os resíduos de aminoácidos, nas posições 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 e 721 estão mutados nos correspondentes resíduos de aminoácidos de AMEP de tipo selvagem de roedor, mas não com o domínio extracelular de AMEP de roedor; (b) um domínio de ligação capaz de se ligar ao domínio extracelular da PAFa quimérica humana, mutada, com uma sequência de aminoácidos, tal como descrito na SEQ ID N2: 448, em que os resíduos de aminoácidos nas posições 144, 185, 186, 229, 267, 273, 274, 278, 284, 301, 328, 329, 331, 335 e 362 estão mutados nos correspondentes resíduos de aminoácidos de PAFa de tipo selvagem de roedor, mas não com o domínio extracelular de PAFa de roedor; (c) um domínio de ligação capaz de se ligar aos quatro domínios de Ig com a SEQ ID N2: 436, um domínio rico de cisteína com a seq ID: 437 ou a cadeia beta de um somínio sema com a seq ID: 438 do domínio extracelular de c-MET; (d) um domínio de ligação capaz de se ligar ao domínio de mucina com a seq ID: 440, aos três domínios semelhantes a FCE com a seq ID: 441 ou o domínio de Sushi/SCR/CCP com a seq ID: 442 do domínio extracelular de TEMI; e (e) um domínio de ligação capaz de se ligar aos três domínios de fibronectina do tipo III, domínio que tem a seq ID: 444 e o domínio L2 com a seq ID: 445 do domínio extracelular de Rl-FCI.A single-chain bispecific antibody comprising a first binding domain capable of binding to an antigenic determinant of CD3 epsilon (CD3s) from human beings and non-chimpanzee primates, wherein the antigenic determinant is part of an amino acid sequence comprised in the group which consists of SEQ ID N2s. 2, 4, 6 and 8 and a second binding domain selected from the group consisting of: (a) a binding domain capable of binding to the human AMEP extracellular domain mutated with an amino acid sequence such as SEQ ID N2. 447, wherein the amino acid residues at positions 140, 169, 191, 308, 334, 339, 344, 624, 626, 716, 717 and 721 are mutated into the corresponding amino acid residues of wild-type rodent AMEP, but not with the extracellular domain of rodent AMEP; (b) a binding domain capable of binding to the mutated human chimeric PAF extracellular domain having an amino acid sequence as described in SEQ ID NO: 448, wherein amino acid residues at positions 144, 185, 186 , 229, 267, 273, 274, 274, 278, 284, 301, 328, 329, 331, 335 and 362 are mutated at the corresponding wild type PAFa amino acid residues of rodent but not with the extracellular domain of rodent PAFα; (c) a binding domain capable of binding to the four Ig domains with SEQ ID NO: 436, a cysteine rich domain having seq ID: 437 or the beta chain of a seventh somnase with SEQ ID: 438 extracellular domain of c-MET; (d) a binding domain capable of binding to the mucin domain having SEQ ID: 440, to the three FCE-like domains with SEQ ID: 441 or the Sushi / SCR / CCP domain having SEQ ID: 442 extracellular domain of TEMI; and (e) a binding domain capable of binding to the three type III fibronectin domains, domain having seq ID: 444 and the L2 domain having sequence ID: 445 of the extracellular domain of R 1 -IFF. 9. Sequência de ácidos nucleicos, caracterizada pelo facto de codificar para uma molécula de anticorpo bi-específico de cadeia única, tal como definido na reivindicação 8.A nucleic acid sequence characterized in that it encodes a single-chain bi-specific antibody molecule as defined in claim 8. 10. Vetor, caracterizado pelo facto de compreender uma sequência de ácidos nucleicos, tal como definido na reivindicação 9.A vector, which comprises a nucleic acid sequence as defined in claim 9. 11. Célula hospedeira caracterizada pelo facto de estar transformada ou transfectada com um vetor definido na reivindicação 10.A host cell characterized in that it is transformed or transfected with a vector defined in claim 10. 12. Processo para a produção de uma molécula de anticorpo biespecifico de cadeia única, de acordo com a reivindicação 8, caracterizado pelo facto de o referido processo compreender a cultura de uma célula hospedeira, definida na reivindicação 11, em condições que permitem a expressão do polipéptido, tal como definido na reivindicação 8 e a recuperação do polipéptido produzido a partir da cultura.A process for the production of a single-chain bispecific antibody molecule according to claim 8, characterized in that said method comprises culturing a host cell as defined in claim 11 under conditions allowing expression of the polypeptide as defined in claim 8 and recovering the polypeptide produced from the culture. 13. Composição farmacêutica, caracterizada pelo facto de compreender uma molécula de anticorpo biespecifico de cadeia única, de acordo com a reivindicação 8 ou por ser produzida de acordo com o processo da reivindicação 12 . Lisboa, 25 de Maio de 2016.A pharmaceutical composition comprising a single chain bispecific antibody molecule according to claim 8 or in that it is produced according to the process of claim 12. Lisbon, May 25, 2016.
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